The Molecular Mechanism Of Malonate-Induced The Assembly Of Cytoplasmic Stress Granules

Objectives: Organism lives in the complicated and changeable environment, and the external stress, such as heat shock, cold stress, oxidative stress, virus infection and UV irradiation, will stimulate eukaryotic cells to adapt and form a more complicated "cellular stress system", including stress granules(SGs), processing bodies(PBs), mitochondrial stress, endoplasmic reticulum stress(ERS) and so on. It’s meaningful to study the functional links among the above cellular defense mechanisms during stress. Accumulating evidence indicates that SGs are associated with the endoplasmic reticulum stress(ERS) and mitochondrial stress. It was reported that thapsigargin, a kind of intracellular ER calcium pump inhibitor, is able to induce the recruitment of TAR DNA binding protein 43(TDP-43) or staufen 1 protein into SGs, and the molecular mechanism of endoplasmic reticulum stress-induceed SGs had been reported. A variety of drugs, such as the inhibitor of electron transport of respiratory chain or uncoupling agent lead to the occurrence of mitochondrial stress. FCCP and paraquat were reported to induce the formation of SGs. However, the underlying molecular mechanism between mitochondrial stress and SGs assembly remains elusive. In the present study, we aimed at further studying the molecular mechanism between mitochondrial stress and SGs assembly.Methods: The research was divided into three parts. PART one:(1)The cell proliferation assay and immunofluorescence(IF) assay were performed to confirm the optimum concentration of malonate-induced SGs formation.(2)The immunofluorescence assay was performed to analysis the cellular co-localization of endogenous SGs marker proteins.(3)The ATP assay, Reactive Oxygen Species assay, mitochondrial membrane potential assay and mitochondrion-selective probes were performed to determine malonate-induced the mitochondrial dysfunction.(4) The IF assay and live-cell imaging assay were performed to study the localization of SGs marker proteins and mitochondrial components. PART two:(1)The sucrose gradient analysis of polysomes was performed to study the association between translation repression and SGs formation.(2)Combination of the Western blotting, si RNA transfection and IF assay were performed to analysis the association between eIF2α and SGs aggregation.(3)Combination of the Western blotting, si RNA transfection and IF assay were performed to study the association between 4EBP1 and SGs aggregation.(4)Combination of the Western blotting, the MAPK inhibitors and IF assay were performed to study the association between MAPK pathway and SGs aggregation. PART three:(1)Cell Titer-Glo? luminescent cell viability assay, Western blotting, IF assay, cell apoptosis assay and cell proliferation assay were performed to study the association between ATP and SGs formation.(2)Reactive Oxygen Species assay, Western blotting, IF assay, cell apoptosis assay and cell proliferation assay were performed to study the association between ROS and SGs formation.Results: PART one:(1)Under malonate stress which drug concentration is between 0 m M and 500 m M, the cell proliferation had no significant difference. We observed small SGs in the cytoplasm at 50 m M of malonate. The maximum size and number of SGs were observed at 100 m M of malonate. Increasing the concentration of malonate, SGs disappeared.(2)The endogenous SGs marker proteins G3 BP co-localizes with TIAR, TIA-1, Hu R proteins at 100 m M of malonate.(3)Compared with control group, 100 m M malonate exposure enhanced ROS formation, reduced mitochondrial membrane potential and the level of ATP. Mitochondria formed an elongated tubular network under normal condition, whereas mitochondria were arranged in circular-shaped clumps upon malonate treatment.(4) Under stress conditions,mitochondria were arranged in circular-shaped clumps and aggregated around nucleus, whereas SGs aggregated around mitochondrial. PART two:(1) The polysome profiles indicated that both arsenite sodium and malonate could induce the polysomes collaspe during stress, compared with control group.(2) Malonate treatment significantly enhanced the expression of phosphorylated eIF2α protein. Upon malonate treatment, the down-regulation of HRI or GCN2 expression attenuated the malonate-induced enhancement of eIF2α phosphorylation level, but not PKR or PERK, compared with scramble si RNA group. The knockdown of HRI or GCN2 via si RNA transfection failed to significantly affect the size of SGs and foci number in the majority of He La cells under malonate-induced mitochondrial stress.(3)Reduced band signal of phosphorylated 4EBP1 was detected in malonate-treated group, compared with the untreated group. When 4EBP1 expression was down-regulated, the size and number of SGs was reduced, compared with scramble si RNA group.(4)Malonate activated three MAPK kinases, p38 MAPK, Erk1/2 and SAPK/JNK. Selective inhibitors of p38, Erk1/2 and SAPK/JNK(SB203580, PD98059 or SP600125, respectively) were used in combination with malonate in order to inhibite the kinase of these pathways. While inhibition of p38, Erk1/2 and SAPK/JNK had no influence on SGs formation. PART three:(1) Compared with malonate treatment group, the size of malonate-induced SGs increases in ATP/malonate group. The foci number in the majority of cells is no significant difference, when ATP is added. When the cells were preincubated with ATP or not, compared with control group, both the treatment of malonate and ATP/malonate could decrease 4EBP1 phosphorylation and increase the eIF2α phosphorylation. However, the phosphorylation levels of 4EBP1 or eIF2α between malonate and ATP/malonate group exhibited no significant difference. Compared with control group, both the treatment of malonate and malonate/ATP could induce the cleavage of caspase 3 protein, suggesting that apoptosis was slightly induced in malonate-treated He La cells, and ATP failed to attenuate the process. ATP has no effect on the proliferation of malonate-treated He La cells.(2)Compared with malonate treatment group, the size and the number of malonate-induced SGs is no significant difference in NAC/malonate group, when ROS scavengers NAC suppresses the enhanced ROS. However, the phosphorylation levels of 4EBP1 or eIF2α between malonate and NAC/malonate group exhibited no significant difference. Compared with control group, NAC has no effect on the the cleavage of caspase 3 protein, apoptosis and proliferation of malonate-treated He La cells.Conclusions: 1)Under malonate stress,SGs marker protein G3 BP co-localizes with SGs related proteins TIAR, TIA-1 and Hu R protein, participating in the cytoplasmic SGs aggregation. Malonate stimulation is able to induce mitochondrial stress. The level of ATP, ROS, mitochondrial membrane potential and the prominent morphological changes in 100 m M malonate-treated He La cells. 2)Compared with control group, the phosphorylation of eIF2α, 4EBP1 and MAPK has changed under stress condition. The 4EBP1 pathway is involved in the malonate-induced SGs aggregation and eIF2α phosphorylation pathway and MAPK pathway appears not to be the major mechanism during the process of malonate-induced SGs assembly. 3)ATP enhances malonate-induced SGs aggregation, but not through 4EBP1 or eIF2α pathway. ATP has no effect on the apoptosis or proliferation of malonate-treated He La cells. NAC has no effect on the size and number of SGs, cell apoptosis and proliferation under malonate stress.